The present invention relates generally to polyketides and polyketide synthases. In particular, the invention pertains to the recombinant production of polyketides using a novel host-vector system. In addition, the invention relates to the combinatorial biosynthesis of polyketides.
Polyketides are a large, structurally diverse family of natural products. Polyketides possess a broad range of biological activities including antibiotic and pharmacological properties. For example, polyketides are represented by such antibiotics as tetracyclines and erythromycin, anticancer agents including daunomycin, immunosuppressants, for example FK506 and rapamycin, and veterinary products such as monensin and avermectin. Polyketides occur in most groups of organisms and are especially abundant in a class of mycelial bacteria, the actinomycetes, which produce various polyketides.
Polyketide synthases (PKSs) are multifunctional enzymes related to fatty acid synthases (FASs). PKSs catalyze the biosynthesis of polyketides through repeated (decarboxylative) Claisen condensations between acylthioesters, usually acetyl, propionyl, malonyl or methylmalonyl. Following each condensation, they introduce structural variability into the product by catalyzing all, part, or none of a reductive cycle comprising a ketoreduction, dehydration, and enoylreduction on the xcex2-keto group of the growing polyketide chain. PKSs incorporate enormous structural diversity into their products, in addition to varying the condensation cycle, by controlling the overall chain length, choice of primer and extender units and, particularly in the case of aromatic polyketides, regiospecific cyclizations of the nascent polyketide chain. After the carbon chain has grown to a length characteristic of each specific product, it is released from the synthase by thiolysis or acyltransfer. Thus, PKSs consist of families of enzymes which work together to produce a given polyketide. It is the controlled variation in chain length, choice of chain-building units, and the reductive cycle, genetically programmed into each PKS, that contributes to the variation seen among naturally occurring polyketides.
Two general classes of PKSs exist. One class, known as Type I PKSS, is represented by the PKSs for macrolides such as erythromycin. These xe2x80x9ccomplexxe2x80x9d or xe2x80x9cmodularxe2x80x9d PKSs include assemblies of several large multifunctional proteins carrying, between them, a set of separate active sites for each step of carbon chain assembly and modification (Cortes, J. et al. Nature (1990) 348:176; Donadio, S. et al. Science (1991) 252:675; MacNeil, D. J. et al. Gene (1992) 115:119). Structural diversity occurs in this class from variations in the number and type of active sites in the PKSs. This class of PKSs displays a one-to-one correlation between the number and clustering of active sites in the primary sequence of the PKS and the structure of the polyketide backbone.
The second class of PKSs, called Type II PKSs, is represented by the synthases for aromatic compounds. Type II PKSs have a single set of iteratively used active sites (Bibb, M. J. et al. EMBO J. (1989) 8:2727;,Sherman, D. H. et al. EMBO J. (1989) 8:2717; Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278).
In contrast, fungal PKSs, such as the 6-methylsalicylic acid PKS, consist of a single multi-domain polypeptide which includes all the active sites required for the biosynthesis of 6-methylsalicylic acid (Beck, J. et al. Eur. J. Biochem. (1990) 192:487-498; Davis, R. et al. Abstr. of the Genetics of Industrial Microorganism Meeting, Montreal, abstr. P288 (1994)).
Streptomyces is an actinomycete which is an abundant producer of aromatic polyketides. In each Streptomyces aromatic PKS so far studied, carbon chain assembly requires the products of three open reading frames (ORFs). ORF1 encodes a ketosynthase (KS) and an acyltransferase (AT) active site; ORF2 encodes a PKS chain length determining factor (CLF); and ORF3 encodes a discrete acyl carrier protein (ACP).
Streptomyces coelicolor produces the blue-pigmented polyketide, actinorhodin. The actinorhodin gene cluster (act), has been cloned (Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462; Malpartida, F. and Hopwood, D. A. Mol. Gen. Genet. (1986) 205:66) and completely sequenced (Fernandez-Moreno, M. A. et al. J. Biol. Chem. (1992) 267:19278; Hallam, S. E. et al. Gene (1988) 74:305; Fernandez-Moreno, M. A. et al. Cell (1991) 66:769; Caballero, J. et al. Mol. Gen. Genet. (1991) 230:401). The cluster encodes the PKS enzymes described above, a cyclase and a series of tailoring enzymes involved in subsequent modification reactions leading to actinorhodin, as well as proteins involved in export of the antibiotic and at least one protein that specifically activates transcription of the gene cluster. Other genes required for global regulation of antibiotic biosynthesis, as well as for the supply of starter (acetyl CoA) and extender (malonyl CoA) units for polyketide biosynthesis, are located elsewhere in the genome.
The act gene cluster from S. coelicolor has been used to produce actinorhodin in S. parvulus. Malpartida, F. and Hopwood, D. A. Nature (1984) 309:462. Bartel et al. J. Bacteriol. (1990) 172:4816-4826, recombinantly produced aloesaponarin II using S. galilaeus transformed with an S. coelicolor act gene cluster consisting of four genetic loci, acti, actIII, actIV and actVII. Hybrid PKSs, including the basic act gene set but with ACP genes derived from granaticin, oxytetracycline, tetracenomycin and frenolicin PKSs, have also been designed which are able to express functional synthases. Khosla, C. et al. J. Bacteriol. (1993) 175:2197-2204. Hopwood, D. A. et al. Nature (1985) 314:642-644, describes the production of hybrid polyketides, using recombinant techniques. Sherman, D. H. et al. J. Bacteriol. (1992) 174:6184-6190, reports the transformation of various S. coelicolor mutants, lacking different components of the act PKS gene cluster, with the corresponding granaticin (gra) genes from S. violaceoruber, in trans.
However, no one to date has described the recombinant production of polyketides using genetically engineered host cells which substantially lack their entire native PKS gene clusters.
The present invention provides for novel polyketides and novel methods of efficiently producing both new and known polyketides, using recombinant technology. In particular, a novel host-vector system is used to produce PKSs which in turn catalyze the production of a variety of polyketides. Furthermore, methods are provided for the combinatorial biosynthesis of polyketide libraries which can be screened for active compounds. Such polyketides are useful as antibiotics, antitumor agents, immunosuppressants and for a wide variety of other pharmacological purposes.
Accordingly, in one embodiment, the invention is directed to a genetically engineered cell which expresses a polyketide synthase (PKS) gene cluster in its native, nontransformed state, the genetically engineered cell substantially lacking the entire native PKS gene cluster.
In another embodiment, the invention is directed to the genetically engineered cell as described above, wherein the cell comprises:
(a) a replacement PKS gene cluster which encodes a PKS capable of catalyzing the synthesis of a polyketide; and
(b) one or more control sequences operatively linked to the PKS gene cluster, whereby the genes in the gene cluster can be transcribed and translated in the genetically engineered cell,
with the proviso that when the replacement PKS gene cluster comprises an entire PKS gene set, at least one of the PKS genes or control elements is heterologous to the cell.
In particularly preferred embodiments, the genetically engineered cell is Streptomyces coelicolor, the cell substantially lacks the entire native actinorhodin PKS gene cluster and the replacement PKS gene cluster comprises a first gene encoding a PKS ketosynthase and a PKS acyltransferase active site (KS/AT), a second gene encoding a PKS chain length determining factor (CLF), and a third gene encoding a PKS acyl carrier protein (ACP).
In another embodiment, the invention is directed to a method for producing a recombinant polyketide comprising:
(a) providing a population of cells as described above; and
(b) culturing the population of cells under conditions whereby the replacement PKS gene cluster present in the cells, is expressed.
In still another embodiment, the invention is directed to a method for producing a recombinant polyketide comprising:
(a) inserting a first portion of a replacement PKS gene cluster into a donor plasmid and inserting a second portion of a replacement PKS gene cluster into a recipient plasmid, wherein the first and second portions collectively encode a complete replacement PKS gene cluster, and further wherein:
i. the donor plasmid expresses a gene which encodes a first selection marker and is capable of replication at a first, permissive temperature and incapable of replication at a second, non-permissive temperature;
ii. the recipient plasmid expresses a gene which encodes a second selection marker; and
iii. the donor plasmid comprises regions of DNA complementary to regions of DNA in the recipient plasmid, such that homologous recombination can occur between the first portion of the replacement PKS gene cluster and the second portion of the replacement gene cluster, whereby a complete replacement gene cluster can be generated;
(b) transforming the donor plasmid and the recipient plasmid into a host cell and culturing the transformed host cell at the first, permissive temperature and under conditions which allow the growth of host cells which express the first and/or the second selection markers, to generate a first population of cells;
(c) culturing the first population of cells at the second, non-permissive temperature and under conditions which allow the growth of cells which express the first and/or the second selection markers, to generate a second population of cells which includes host cells which contain a recombinant plasmid comprising a complete PKS replacement gene cluster;
(d) transferring the recombinant plasmid from the second population of cells into the genetically engineered cell described above to generate transformed genetically engineered cells; and
(e) culturing the transformed genetically engineered cells under conditions whereby the replacement PKS gene cluster present in the cells is expressed.
In a further embodiment, the invention is drawn to a method for preparing a combinatorial polyketide library comprising:
(a) providing a population of vectors wherein the vectors comprise a random assortment of polyketide synthase (PKS) genes, modules, active sites, or portions thereof and one or more control sequences operatively linked to said genes;
(b) transforming a population of host cells with said population of vectors;
(c) culturing said population of host cells under conditions whereby the genes in said gene cluster can be transcribed and translated, thereby producing a combinatorial library of polyketides.
In still another embodiment, the invention is drawn to a method for producing a combinatorial polyketide library comprising:
a) providing one or more expression plasmids containing a random assortment of 1 or more first modules of a modular PKS gene cluster wherein the expression plasmids express a gene which encodes a first selection marker;
b) providing a pool of donor plasmids containing a random assortment of second modules of a modular PKS gene cluster wherein the donor plasmids express a gene which encodes a second selection marker and further wherein the donor plasmids comprise regions of DNA complementary to regions of DNA in the expression plasmids, such that homologous recombination can occur between the first and second modules;
c) transforming the expression plasmids and the donor plasmids into a first population of host cells to produce a first pool of transformed host cells;
d) culturing the first pool of transformed host cells under conditions which allow homologous recombination to occur between the first and second modules to produce recombined plasmids comprising recombined PKS gene cluster modules;
e) transferring the recombined plasmids into a second population of host cells to generate a second pool of transformed host cells; and
f) culturing the second pool of transformed host cells under conditions whereby the combinatorial polyketide library is produced.
In yet another embodiment, the invention is directed to a polyketide compound having the structural formula (I) 
wherein:
R1 is selected from the group consisting of hydrogen and lower alkyl and R2 is selected from the group consisting of hydrogen, lower alkyl and lower alkyl ester, or wherein R1 and R2 together form a lower alkylene bridge optionally substituted with one to four hydroxyl or lower alkyl groups;
R3 and R5 are independently selected from the group consisting of hydrogen, halogen, lower alkyl, lower alkoxy, amino, lower alkyl mono- or di-substituted amino and nitro;
R4 is selected from the group consisting of halogen, lower alkyl, lower alkoxy, amino, lower alkyl mono- or di-substituted amino and nitro;
R6 is selected from the group consisting of hydrogen, lower alkyl, and xe2x80x94CHR7xe2x80x94(CO)R8 where R7 and R8 are independently selected from the group consisting of hydrogen and lower alkyl; and
i is 1, 2 or 3.
In another embodiment, the invention related to novel polyketides having the structures 
In another embodiment, the invention is directed to a polyketide compound formed by catalytic cyclization of an enzyme-bound ketide having the structure (II) 
wherein:
R11 is selected from the group consisting of methyl, xe2x80x94CH2(CO)CH3 and xe2x80x94CH2(CO)CH2(CO)CH3;
R1 is selected from the group consisting of xe2x80x94Sxe2x80x94E and xe2x80x94CH2(CO)xe2x80x94Sxe2x80x94E, wherein E represents a polyketide synthase produced by the genetically engineered cells above; and
one of R13 and R14 is hydrogen and the other is hydroxyl, or R13 and R14 together represent carbonyl.
In still another embodiment, the invention is directed to a method for producing an aromatic polyketide, comprising effecting cyclization of an enzyme-bound ketide having the structure (II), wherein cyclization is induced by the polyketide synthase.
In a further embodiment, the invention is directed to a polyketide compound having the structural formula (III) 
wherein R2 and R4 are as defined above and i is 0, 1 or 2.
In another embodiment, the invention is directed to a polyketide compound having the structural formula (IV) 
wherein R2, R4 and i are as defined above for structural formula (III).
In still anther embodiment, the invention is directed to a polyketide compound having the structural formula (V) 
wherein R2, R4 and i are as defined above for structural formula (III).
These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.